Angiotensin converting enzyme (peptidyl dipeptidase A, EC 3.4.15.1, ACE) is a membrane-anchored dipeptidyl carboxypeptidase that is essential for blood pressure regulation and electrolyte homeostasis via the renin-angiotensin-aldosterone system. The enzyme is a zinc metalloprotease that converts the inactive peptide angiotensin I to angiotensin II, a potent vasoconstrictor. ACE, like many diverse membrane-bound ectoproteins, is released from the membrane by a membrane protease or secretase (1). An understanding of this cleavage-secretion mechanism is important for the development of therapeutic strategies to address the different pathologies caused by defects in the function of the secretase. Substrate determinants that specify cleavage by secretases remain incompletely characterised, but may include the physico-chemical properties of the juxtamembrane (“stalk”) sequence or unidentified recognition motifs of the stalk or the extracellular domain. Cleavage of ACE occurs in the stalk sequence and the solubilizing protease is constrained topologically, in terms of the number of residues between the cleavage site and the proximal extracellular domain of ACE. However, the ACE secretase appears to be remarkably versatile in terms of its substrate specificity (2,3).
The active sites of ACE and carboxypeptidase A, a prototypic zinc metalloprotease, are understood to be very similar and this similarity is exploited in the design of the first generation of ACE inhibitors. The clinical success of these inhibitors—such as captopril and enalapril—in the treatment of hypertension and congestive heart failure is well established. However, the side effects such as persistent cough which effects up to 20% of patients and angioedema which is less common, together with limitations such as their contraindication in patients with impaired renal function and decreased efficacy in patients with low-renin hypertension, underscore the need for more specific and selective inhibitors.
There are two isoforms of ACE that are transcribed from the same gene in a tissue specific manner. In somatic tissues it exists as a glycoprotein composed of a single large polypeptide chain of 1277 amino acids whereas the germinal form is synthesised as a lower molecular mass isozyme and is thought to play a role in sperm maturation and the binding of sperm to the oviduct epithelium. The somatic form consists of two domains (N- and C-domain), each containing an active site with a conserved HEXXH zinc-binding motif and a glutamate some 24 residues downstream which forms the third zinc ligand (Williams et al., 1994). The two domains differ in their substrate specificities; inhibitor and chloride activation profiles; and physiological functions. There are two N-domain-specific substrates: the hemoregulatory peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) which controls hematopoietic stem cell differentiation and proliferation; and the bradykinin potentiating peptide angiotensin-(1-7). On the other hand, both active sites catalyse the hydrolysis of angiotensin I and the vasodilator bradykinin with similar efficiency. However, inhibition of the N-domain with a phosphinic peptide RXP407 has no effect on blood pressure regulation (Junot et al., 2001) and, furthermore, expression of the N-domain only, in transgenic mice produced a phenotype similar to the ACE knockout mice (Esther et al., 1997). Thus, the C-domain appears to be necessary and sufficient for controlling blood pressure and cardiovascular function. Testis ACE (tACE) is identical to the C-terminal half of somatic ACE, except for a unique 36-residue sequence constituting its amino terminus, thus this isoform is selected for initial efforts to obtain a three-dimensional structure.
The cDNA sequence of human testicular ACE has been described (Ehlers et al. (1989) Proc. Nat. Acad. Sci. 86: 7741-7745) and the predicted protein consists of a 732-residue preprotein including a 31-residue signal peptide. The mature polypeptide has a molecular weight of 80,073 (unglycosylated form).
Despite the pivotal role of ACE, there have been no reports disclosing that suitable crystals have been or could be obtained for this enzyme and so the X-ray crystallographic analysis of such proteins has been impossible.